The Effects of Titanium Dioxide Nanoparticles on Osteoblasts Mineralization: A Comparison between 2D and 3D Cell Culture Models.

Although several studies assess the biological effects of micro and titanium dioxide nanoparticles (TiO2 NPs), the literature shows controversial results regarding their effect on bone cell behavior. Studies on the effects of nanoparticles on mammalian cells on two-dimensional (2D) cell cultures display several disadvantages, such as changes in cell morphology, function, and metabolism and fewer cell-cell contacts. This highlights the need to explore the effects of TiO2 NPs in more complex 3D environments, to better mimic the bone microenvironment. This study aims to compare the differentiation and mineralized matrix production of human osteoblasts SAOS-2 in a monolayer or 3D models after exposure to different concentrations of TiO2 NPs. Nanoparticles were characterized, and their internalization and effects on the SAOS-2 monolayer and 3D spheroid cells were evaluated with morphological analysis. The mineralization of human osteoblasts upon exposure to TiO2 NPs was evaluated by alizarin red staining, demonstrating a dose-dependent increase in mineralized matrix in human primary osteoblasts and SAOS-2 both in the monolayer and 3D models. Furthermore, our results reveal that, after high exposure to TiO2 NPs, the dose-dependent increase in the bone mineralized matrix in the 3D cells model is higher than in the 2D culture, showing a promising model to test the effect on bone osteointegration.

The role of macrophages in fracture healing: a narrative review of the recent updates and therapeutic perspectives.

Objective: This review addresses the latest advances in research on the role of macrophages in fracture healing, exploring their relationship with failures in bone consolidation and the perspectives for the development of advanced and innovative therapies to promote bone regeneration. Background: The bone can fully restore its form and function after a fracture. However, the regenerative process of fracture healing is complex and is influenced by several factors, including macrophage activity. These cells have been found in the fracture site at all stages of bone regeneration, and their general depletion or the knockdown of receptors that mediate their differentiation, polarization, and/or function result in impaired fracture healing. Methods: The literature search was carried out in the PubMed database, using combinations of the keywords "macrophage", "fracture healing, "bone regeneration", and "bone repair". Articles published within the last years (2017-2022) reporting evidence from in vivo long bone fracture healing experiments were included. Conclusions: Studies published in the last five years on the role of macrophages in fracture healing strengthened the idea that what appears to be essential when it comes to a successful consolidation is the right balance between the M1/M2 populations, which have different but complementary roles in the process. These findings opened promising new avenues for the development of several macrophage-targeted therapies, including the administration of molecules and/or biomaterials intended to regulate macrophage differentiation and polarization, the local transplantation of macrophage precursors, and the use of exosomes to deliver signaling molecules that influence macrophage activities. However, more research is still warranted to better understand the diversity of macrophage phenotypes and their specific roles in each step of fracture healing and to decipher the key molecular mechanisms involved in the in vivo crosstalk between macrophages and other microenvironmental cell types, such as endothelial and skeletal stem/progenitor cells.

Establishing a Diaphyseal Femur Fracture Model in Mice.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

Vertical femoral neck fractures in young adults: a closed fixation strategy using a transverse cancellous lag screw.

Vertical femoral neck fractures (Pauwels type III classification) in young adults generally occur as a consequence of high-energy trauma and are frequently seen in association with multiple injuries. Considering the controversies regarding the optimal fixation for this fracture, our aim was to evaluate the clinical outcome of a closed fixation strategy for vertical femoral neck fractures in young adults using two parallel and one transverse cancellous lag screws. This was a single-surgeon, prospective study including 20 young adults with average age of 38.75 years (range 18-59 years) with a high-energy Pauwels III femoral neck fracture. Closed reduction and internal fixation with three cancellous lag screws were performed. The first screw was inserted crosswise to avoid further shear forces. Second and third parallel screws were placed above the lesser trochanter and centrally on the greater trochanter, respectively. Clinical outcomes were assessed by comparing postoperative and final follow-up radiographs 24 months post-injury. Eleven patients had an isolated vertical femoral neck fracture. Of these, five had further femoral neck comminution. Nine patients had an associated ipsilateral femoral shaft fracture. All fractures were displaced at the time of the first radiological evaluation. Closed reduction quality was considered excellent or good in 15 patients. After 24 months, bone union was achieved in 16 cases. Osteonecrosis of the femoral head developed in association with two fractures, and a nonunion developed in association with two fractures. We conclude that vertical high-energy femoral neck fractures can be treated successfully with internal fixation with two parallel cancellous lag screws positioned above the lesser trochanter and a third screw inserted centrally on the greater trochanter at an angle perpendicular to the fracture line.

Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes.

OBJECTIVE: This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. METHODS: Bovine tendons were used for preparation of the extract and were stored at -80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. RESULTS: The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. CONCLUSION: Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes.

OBJETIVO: O estudo avalia a diferenciação in vitro das células mesenquimais isoladas do estroma da medula óssea em tenócitos após tratamento com extrato de tendão bovino. MÉTODOS: Tendões bovinos foram usados para confecção do extrato e estocados a −80 °C. Células mesenquimais do estroma da medula óssea (BMSCs) de três doadores foram usadas para os testes de citotoxicidade por MTT e diferenciação celular por qPCR. RESULTADOS: Os dados mostram que células mesenquimais do estroma da medula óssea tratadas por até 21 dias em presença do extrato de tendão bovino diluído em concentrações crescentes (1:10, 1:50 e 1:250) promovem a ativação da expressão de biglican, colágeno tipo I e fibromodulina. CONCLUSÃO: Nossos resultados mostram que o extrato de tendão bovino é capaz de promover a diferenciação das BMSCs em tenócitos.

Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes / Células mesenquimais do estroma da medula óssea tratadas com extrato de tendão bovino adquirem o fenótipo de tenócitos maduros

This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. METHODS: Bovine tendons were used for preparation of the extract and were stored at -80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. RESULTS: The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. CONCLUSION: Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes.

O estudo avalia a diferenciação in vitro das células mesenquimais isoladas do estroma da medula óssea em tenócitos após tratamento com extrato de tendão bovino. MÉTODOS: Tendões bovinos foram usados para confecção do extrato e estocados a -80 °C. Células mesenquimais do estroma da medula óssea (BMSCs) de três doadores foram usadas para os testes de citotoxicidade por MTT e diferenciação celular por qPCR. RESULTADOS: Os dados mostram que células mesenquimais do estroma da medula óssea tratadas por até 21 dias em presença do extrato de tendão bovino diluído em concentrações crescentes (1:10, 1:50 e 1:250) promovem a ativação da expressão de biglican, colágeno tipo I e fibromodulina. CONCLUSÃO: Nossos resultados mostram que o extrato de tendão bovino é capaz de promover a diferenciação das BMSCs em tenócitos.

Late adherent human bone marrow stromal cells form bone and restore the hematopoietic microenvironment in vivo.

Bone marrow stromal cells (BMSCs) are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term "stem cell," this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells) and those that did not adhere by three days but did by six days (L-cells). Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiation in vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stroma in vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics.

Heterologous mesenchymal stem cells successfully treat femoral pseudarthrosis in rats.

BACKGROUND: This study evaluated the effectiveness of treating pseudarthrosis in rats by using bone marrow cell suspensions or cultures of bone marrow mesenchymal stromal cells METHODS: Thirty-eight specific pathogen-free (SPF) animals were randomly assigned to four groups: Group 1, Control, without surgical intervention; Group 2 (Placebo), experimental model of femoral pseudarthrosis treated only with saline solution; Group 3, experimental model of femoral pseudarthrosis treated with heterologous bone marrow cells suspension; Group 4, experimental model of femoral pseudarthrosis treated with cultures of heterologous mesenchymal stromal cells from bone marrow. When pseudarthrosis was confirmed by simple radiological studies, digital radiography and histopathology after a 120-day postoperative period, Groups 2, 3 and 4 were treated as above. At 30, 60 and 90 days after the treatment, all animals were evaluated by simple radiological studies, and at the end of the experiment, the animals were assessed by computed axial tomography and anatomopathological and histomorphometric examinations. RESULTS: Injected cells were detected in the areas affected by pseudarthrosis using scintigraphy within the first 24 hours after their administration. After 60 days, the animals of Group 3 showed callus formation while the animals of Group 4 presented periosteal reaction and had some consolidated areas. In contrast, Group 2 showed a predominance of fibro-osteoid tissue. After 90 days, bone consolidation and remodeling was observed in all animals from Group 3 whereas animals from Group 4 exhibited partial consolidation and those ones from Group 2 persisted with pseudarthrosis. CONCLUSION: The treatment with heterologous bone marrow cells suspension proved to be effective in the treatment of pseudarthrosis whereas cultures of heterologous bone marrow mesenchymal stromal cells did not show the same potential to aid bone healing.